Endo-beta-galactosidase from Escherichia freundii which we have purified and characterized has been useful for structural and immunochemical analysis of cell surface antigens such as Ii, ABH, F9 and SSEA-1. Further development of endo-beta-galactosidase will provide an indispensible tool in the analysis of the role of cell surface carbohydrate chains in differentiation and tumorigenicity. Expecting that endo-beta-galactosidase from different origins has substrate specificities different from each other, endo-beta-galactosidases of various bacteria will be studied. Endo-betagalactosidase found in Diplococcus pneumoniae and Bacterioides fragilis has been purified and characterized. The former enzyme is found to be unique in that the enzyme hydrolyzes the lactosaminoglycan (LAG) by stepwise endoglycosidic action. The latter was found to be similar to E. freundii enzyme. Since antigens Ii, F9 and SSEA-1 are specifically expressed at different stages of mouse embryonal differentiation, glycoconjugate profiles of cell surfaces were analyzed by using mouse teratocarcinoma cell lines as a model system. "Nullipotent" embryonal carcinoma cell line (F9) was stimulated to differentiate into parietal endoderm in the presence of retinoic acid and dibutyryl cyclic AMP, or into visceral endoderm in the presence of retinoic acid. Surface glycoconjugates of each stage of cells were analyzed for whether each component has polylactosamino structure which is characterized by susceptibility to endo-beta-galactosidase. The results obtained by surface labeling and endo-beta-galactosidase digestion were consistent with the antigenic expression of these cells. In addition, carbohydrate antigens on cell surfaces will be isolated as immune complexes with specific antibodies using protein A-Sepharose columns. The structure of their carbohydrate moiety will be characterized by endo-beta-galactosidase digestion as well as by hydrazinolysis and nitrous depolymerization. These studies will inform us whether structural changes of lactosaminoglycan have occurred on specific proteins or on all surface glycoproteins, and will provide us with the molecular basis of cell surface changes during embryonal differentiation.